Chromatography

Physical separation method based on the differential

migration of analytes in a mobile phase as they move
along a stationary phase.

Mechanisms of Separation:
•Partitioning
•Adsorption
•Exclusion
•Ion Exchange
•(Affinity)

Classification of Chromatography

Column Chromatography – the stationary phase is held

in a narrow tube through which the mobile phase is forced
under pressure or by gravity.

Planar Chromatography – the stationary phase is supported

on a flat plate or the interstices of a paper and the mobile
phase moves through the stationary phase by capillary action
or by gravity.

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 Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

Chromatographic Separations
Based on the distribution (partitioning) of the solutes
between the mobile and stationary phases, described
by partition coefficient, K:
K = Cs/Cm
where Cs is the solute concentration in the stationary
phase and Cm is its concentration in the mobile phase.

Distribution isotherms (Cs vs. Cm) and

the expected peak shape resulting from
these isotherms. Ideally, Cs is proportional
to Cm.

Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.

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 Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.

Chromatographic Efficiency
Theoretical plates, borrowed from distillation theory. At each
plate, it is assumed that an equilibrium of the solute between
the mobile and stationary phases takes place. Solute movement
is viewed as a series of stepwise transfers from one plate to
the next.

Height equivalent to a theoretical plate (H or HETP) and number

Of theoretical plates (N) are measures of column efficiency:
L = NH
where L is the column length.

As H decreases, efficiency increases since there will be more

equilibrations in along the column.

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 Theoretical plates
N = 16 (tr/W)2 = 5.54 (tr/W1/2)2

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

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 Van Deemter Equation:

H = A + B/u + Csu + Cmu

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

Example of “A” term of van Deemter

Equation, depicting peak broadening
due to multiple flow paths. Note that
in capillary columns, this term reduces
to zero.

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

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Other peak-broadening factors
Longitudinal diffusion (B term)
•Due to diffusion away from (concentrated) center of flow.
•Only significant in GC, not LC.
•Directly proportional to diffusion of analyte in mobile phase.
•Inversely proportional to linear velocity of mobile phase.

Mass Transfer (C terms)

•Related to diffusion of analyte between phases.
•Inversely proportional to diffusion.

Van Deemter plot, showing contributions of A, B, and C terms to

column efficiency, H.

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

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Measures of chromatographic performance
tr = retention time of analyte

tm = retention time of unretained peak

k’ = capacity factor, widely used to characterize performance

k’ = KVs/Vm or k’ = (tr-tm)/tm = t’r/tm

K = βk’ where β is phase ratio

Measures of chromatographic performance

α) is a measure of the ability of a column to

Selectivity factor (α
resolve two solutes.

α = KB/KA = k’B/k’A

where A is the first peak and B is the second peak.

α is always greater than one.

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Measures of chromatographic performance
Resolution (Rs) is the separation of two peaks.

Rs = [(tr)B – (tr)A]/W (assuming peaks are of equal width)

α-1)/α
= [N1/2/4] [(α α] [k’B/(1+k’B)]

α/(α
N = 16Rs2 [α α-1)]2 [(1+k’B)/k’B]2

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

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 Improving Resolution
Improve efficiency (narrower peaks)
Or
Improve separation (peaks farther apart)

Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.

Chromatography Elution Problem

Efficiency decreases for later eluting solutes.

Improve by increasing the mobile phase “strength” during

The course of the chromatographic run (i.e., “programming”)
•GC = Temperature Programming
•LC = Gradient Elution
•SFC = Pressure (density) Programming, some gradient elution

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

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Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

Examples of methods to quantitatively measure peak area.

Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.

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Summary of Chromatography Equations
Partition Coefficient (K) is related to the stationary phase (β β ) and mobile phase (via
retention time) K = Cs/Cm K = β k’
Capacity Factor (k’) reflects retention of solute on column
k’ = (tr-tm)/tm = t’r/tm
Column efficiency is defined by height equivalent to a theoretical plate (plate height, H)
or plate number (N). More plates or smaller plate height means greater efficency.
plate number can be determined from retention time and peak width.
L = NH N = 16 (tr/W)2 = 5.54 (tr/W1/2)2
Plate height (H) is related to mobile phase linear velocity (υ υ) and diffusion. Diffusion
explains why capillary columns are more efficient than packed columns, GC is more
efficient than LC, and capillary (open-tubular) columns are not used in LC often.
H = A + B/υ υ + Csυ + Cmυ
Resolution (Rs) describes how well separated two peaks are and can be measured from
retention time and peak width. It is related to the square root of efficiency (e.g., if
you double the column length, resolution only improves by the square root of two),
selectivity, and retention.
α-1)/α
Rs = [(tr)B – (tr)A]/W = [N1/2/4] [(α α] [k’B/(1+k’B)]
Selectivity is the measure of how well one peak is retained in preference to another.
α = KB/KA = k’B/k’A

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